Acute and long-term effects of cannabinoids on hypertension and kidney injury

Cannabinoids and their endogenous and synthetic analogs impact blood pressure and contribute to the incidence of hypertension. It was previously reported that the endocannabinoid system plays an important role in developing hypertension; however, it was also shown that cannabinoids elicit profound hypotension associated with hemorrhagic, cardiogenic, and endotoxic shock. This study aimed to test acute and chronic effects of an endogenous ligand of cannabinoid receptor anandamide (AEA) on blood pressure and kidney injury in vivo in conscious Dahl salt-sensitive (SS) rats. We demonstrated that acute i.v. bolus administration of a low or a high doses (0.05 or 3 mg/kg) of AEA did not affect blood pressure for 2 h after the injection in Dahl SS rats fed a normal salt diet (0.4% NaCl). Neither low nor high doses of AEA had any beneficial effects on blood pressure or kidney function. Furthermore, hypertensive rats fed a HS diet (8% NaCl) and chronically treated with 3 mg/kg of AEA exhibited a significant increase in blood pressure accompanied by increased renal interstitial fibrosis and glomerular damage at the late stage of hypertension. Western blot analyses revealed increased expression of Smad3 protein levels in the kidney cortex in response to chronic treatment with a high AEA dose. Therefore, TGF-β1/Smad3 signaling pathway may play a crucial role in kidney injury in SS hypertension during chronic treatment with AEA. Collectively, these data indicate that prolonged stimulation of cannabinoid receptors may result in aggravation of hypertension and kidney damage.

www.nature.com/scientificreports/ Cannabinoids and their endogenous and synthetic analogs were implicated in the regulation of blood pressure. Previous studies revealed that short-term administration of endocannabinoids had some beneficial effects on blood pressure [30][31][32] . A number of studies describe that cannabinoids and their endogenous and synthetic analogs exert hypotensive and cardiodepressor effects by complex mechanisms involving direct and indirect effects on myocardium and vasculature 33 . In studies in conscious humans, an acute dose of THC elicited dose-related tachycardia without altering blood pressure 34 . Chronic use of cannabinoids in normotensive men elicit a longlasting decrease in blood pressure and heart rate 35 . In anesthetized rats acute administration of cannabinoids, including AEA, produced hypotension and bradycardia and a dose-dependent drop in arterial pressure 30,36,37 . A separate report showed that in normotensive conscious rats AEA induced a transient increase in the mean arterial blood pressure 38 . Therefore, it became apparent that complex cardiovascular actions of AEA are not consistent and need to be further investigated.
Herein, we used Dahl salt-sensitive (SS) rats to investigate blood pressure changes and kidney function in response to acute and chronic treatment with AEA. The objective of the present study was to perform a detailed characterization of the effects of AEA in Dahl SS rats on a normal salt diet or a high salt diet during the development of salt-induced hypertension. We demonstrate that 1) acute injection of AEA did not affect blood pressure in conscious Dahl SS rats; 2) chronic treatment with a low dose of AEA (0.05 mg/kg) did not modulate blood pressure or kidney function during the development of SS hypertension; 3) a high dose of AEA (3 mg/kg daily) led to a significant aggravation of hypertension and profound renal damage after chronic administration; 4) the negative effect of chronic treatment with AEA was correlated with the upregulated Smad3 intracellular pathway. Collectively, these data provide novel insight into the chronic effects of AEA on developing salt-induced hypertension and kidney damage.

Results
Acute injection of anandamide does not change mean arterial pressure in Dahl SS rats on a normal salt diet. To test the acute effects of AEA we used a single i.v. bolus injection of the drug to Dahl SS rats fed a normal salt diet (NS). The drug was used at a low (Fig. 1A,B) or a high doses (Fig. 1C,D) according to previously reported studies [37][38][39] . Mean arterial pressure (MAP) was monitored through the intraarterial catheter connected to a pressure transducer and averaged every 15 min for two hours following the drug injections. We did not observe acute effects of AEA on MAP at either a low or high doses (Fig. 1).
Evaluation of the effect of chronic treatment with Anandamide on the development of blood pressure and kidney injury in Dahl SS rats on a high salt diet. To determine the effects of chronic exposure to AEA on blood pressure and renal damage during the development of SS hypertension, we used Dahl SS rats fed a high salt (HS) diet. The experimental protocol is shown in Supplementary Figure S1A. The dietary sodium content was changed from NS to HS, which caused profound elevation of blood pressure in rats accompanied by the rapid development of renal damage. To test the effect of chronic AEA exposure, animals were administered 0.05 mg/kg i.v. bolus injections of AEA or vehicle daily from the start of a HS protocol. Chronic treatment with a low dose of AEA exhibited a consistent trend to increase MAP at the second week of the HS  www.nature.com/scientificreports/ protocol ( Fig. 2A). We found no significant differences in the development of albuminuria (Fig. 2B) or creatinine clearance (Fig. 2C) between vehicle-treated and AEA-treated groups. Other renal function parameters like diuresis (Fig. 2D), urine and blood electrolytes, and glucose (Tables 1 and 2) were not different between the groups. The treatment did not affect total body weight (TBW, Fig. 2E), kidney weight (2KW, Fig. 2F), and kidney to body weight ratio (2 K/TBW, Fig. 2G). During the HS challenge in Dahl SS rats, the renal lesion was characterized by hyaline (protein) casts and cortical fibrosis, which were highly present in both groups. Figures 3A,C show a summarized analysis of kidney interstitial fibrosis and medullary casts. Figures 3B,D display representative images of medullary and cortical areas of both groups. Qualitative and statistical comparisons of glomerular injury between the two groups suggesting that the small dose of AEA did not promote any significant changes in the glomeruli injury score are shown in Fig. 3E.  www.nature.com/scientificreports/ In the following experiments, we used a high dose of AEA. The details of the protocol are demonstrated in Supplementary Fig. S1. Blood and urinary electrolyte analyses were summarized in Tables 3 and 4. Significant changes in urinary fractional excretion of calcium during chronic cannabinoid exposure by day 7 of HS challenge was detected. However, when we evaluated urinary ion excretion per day, we did not see differences (Supplementary Tables S1-2). Plasma creatinine remained unchanged between these groups ( Supplementary Fig. S1B, C). In contrast to experiments with a low dose, i.v. administration of a high dose of AEA induced a significant elevation in MAP at the late stage of hypertension (Fig. 4A). Albuminuria, creatinine clearance, and diuresis remained at the same level between the vehicle and AEA-treated groups ( Fig. 4B-D). Both groups had no difference in body and kidney weights ( Fig. 4E-G).
During the development of salt-induced hypertension, a high dose of AEA promoted additional renal damage, reflected by an increase in interstitial fibrosis and worsened glomeruli damage score (Fig. 5A,E). Representative Table 2. Blood electrolytes and glucose analysis of SS rats during chronic experiment with or without treatment with the 0.05 mg/kg of AEA within 14 days on HS. 7DHS, 14DHS -7 days and 14 days on HS (8% NaCl). Data are presented as means ± SE. n ≥ 7 rats for each group; t-test *P < 0.05.  Chronic treatment with a high dose of AEA produced substantial activation of pro-fibrotic Smad3 pathway. To evaluate downstream regulators of AEA-mediated intracellular pathway, we tested β-arrestin 1 expression. Chronic treatment with either dose of AEA revealed no changes neither in protein levels of β-arrestin 1 ( Supplementary Fig. S3) nor its phosphorylated form ( Supplementary Fig. S4). We also evaluated effects of AEA on extracellular signal-regulated kinase (ERK1/2), c-Jun N-terminal kinase (JNK) and their phosphorylated isoforms. However, neither ERK1/2 nor JNK1/2/3 protein levels changed after AEA treatment ( Supplementary Fig. S4). A high AEA dose led to a significant suppression of the antioxidant transcription factor Nuclear factor E2 related factor-2 (Nrf2) mediated intracellular pathway, which is demonstrated by the decrease in Nrf2 and increase in Smad3 expression (Fig. 6). Interestingly, we did not detect significant changes in TGF-β1 expression in our studies (see Supplementary Fig. S4).

Discussion
This study aimed to test acute and chronic effects of an endogenous ligand of cannabinoid receptors AEA on blood pressure and kidney injury in vivo in conscious Dahl SS rats. AEA had no effects on blood pressure in Dahl SS rats on a NS diet. However, prolonged exposure to a high dose of AEA led to the aggravation of SS hypertension and altered kidney functions. AEA could potentially regulate sodium transport at the loop of Henle, where it blocks the Na + /H + exchanger and the NKCC cotransporter 6 . Renal intramedullary infusion of AEA induced urinary sodium excretion through metabolism of AEA to prostamides by COX-2 14,40 . Therefore, in our study, a high dose of AEA induced elevated fractional excretion of calcium only. We identified a significant progression of hypertension in a group treated with a high dose of AEA. The effects on blood pressure were accompanied by a prominent increase in renal interstitial fibrosis and glomerular damage.CB1 receptors are known to play a pivotal role in developing fibrosis in many organs, including liver and kidneys 18   www.nature.com/scientificreports/ decreased the development of fibrosis in mice during unilateral ureteral obstruction, suggesting the role of CB1 in the development of renal fibrosis 41,43,44 . Thus, treatment with the CB1 receptor antagonist was associated with a significant reduction of kidney injury markers (TIMP-1, KIM-1), in agreement with the substantial decrease of fibrosis in the liver 41,43 . In experimental type 1 diabetes, the CB1 receptor was shown to be overexpressed in podocytes. CB1 receptor blockade ameliorated albuminuria most likely by prevention of nephrin, podocin, and ZO-1 loss 45 . Protein expression of CB1 was significantly enhanced in kidney biopsies of patients with IgA nephropathy, diabetes, and acute interstitial nephritis 44 . Several groups elucidated the role of CB2 receptors in the pathogenesis of renal fibrosis [46][47][48][49][50] . Contrary to CB1 receptors, CB2 deletion exacerbated tissue damage by increasing inflammatory, oxidative, and fibrotic processes not only in the kidney but also liver and skin [51][52][53] . CB2 inhibition or pharmacological blockade promotes cardiac, liver, and skin fibrosis, while CB2 agonists decrease fibrogenesis 52,54,55 . CB2 agonist SMM-295 was shown to promote renal vasodilatation by activating vascular and nonvascular CB2 receptors and could potentially be used to treat renal injuries that impact renal blood flow dynamics 56 and AKI 57. Different studies showed that CB2 receptor activation exerted anti-inflammatory activity and reduced oxidative/nitrative tissue injury both in liver and kidneys [58][59][60][61] . Specific CB2 agonist AM1241 reduced albuminuria by preventing loss of podocyte proteins 48,56,57 . CB2 antagonist SR144528 alone aggravated the development of kidney fibrosis while CB2 agonist JWH133 was shown to diminish the development of renal fibrosis during unilateral ureteral obstruction 44 . Barutta et al. revealed that deletion of CB2 receptors during diabetic nephropathy worsened kidney function in a CB2 knockout mice model 49 . Absence of CB2 receptors on resident glomerular cells had a major role in the progression of diabetic nephropathy, most likely due to enhanced CB1 signaling 49 . A separate report by Zhou et al. proposed that the β-arrestin 1-mediated CB2/β-catenin pathway is involved in the development of renal fibrotic injuries 47 . CB2 expression was closely correlated with the progression of kidney fibrosis and was accompanied by the activation of β-catenin. However, this particular signaling pathway needs additional investigation. In the current study we tested several antibodies to detect CB1 and CB2 protein expression (data not shown), but all of them lacked specificity and were therefore unreliable. AEA-induced blood pressure elevation could be developed through CB1-mediated pressor response. A CB1 receptor inverse agonist was shown to slightly increase and prolong the pressor effect of AEA, suggesting that this pressor effect is partially masked by peripheral CB1 receptors, leading to an inhibition of noradrenaline release 62 . AEA content increased in response to blood pressure elevation in a mid-brain region in Sprague Dawley rats and significantly prolonged the baroreflex-mediated inhibition of the renal sympathetic nerve, leading to prolonged CB1 receptor activation 63 . Some conclusions indicated that ventral medial prefrontal cortex CB1 and TRPV1 receptors could facilitate the cardiac baroreflex activity by either stimulating or blocking NMDA activation and  (2KW, F), and 2 kidneys to body weight ratio (2 K/TBW, G) changes in these groups. For the data sets, urine samples were collected for 24 h. n ≥ 7 rats per group. Data were analyzed using one-way ANOVA followed by Holm-Sidak post hoc test. www.nature.com/scientificreports/ NO synthesis 64 . Dahl SS rats, used in the current research, are known to be associated with baroreflex control system dysfunction 65 . Similar to the spontaneously hypertensive rats, afferent baroreceptor nerve fiber in the SS rat is characterized by elevated pressure thresholds and reduced pressure sensitivity 66 . The alternative pathways of AEA-mediated blood pressure response, including the interplay between the renin-angiotensin system and the ECS, suggest that AEA regulates vascular contraction caused by Ang II. Renin-angiotensin system-ECS interactions may contribute to the enhanced vascular reactivity in early stages of hypertensive pregnancy 67 . AEA reduced Ang II-mediated contraction in a CB1-independent manner 67 . AEA caused a brief rise in blood pressure through renal and mesenteric vasoconstriction after administration to conscious Sprague-Dawley rats, and the CB1 receptor antagonist did not affect any of those cardiovascular effects 68 . Alternatively, eicosanoids can also influence vascular and renal mechanisms of blood pressure regulation 69 . AEA is converted into AA by the fatty acid amide hydrolase (FAAH), which is then metabolized by P450s to vasoactive eicosanoids 26,44,70,71 . This pathway may also affect AEA function. Indeed, inhibition of FAAH increased the bioavailability of AEA and dramatically reduced blood pressure in hypertensive rats 30 . The percentage of glomeruli within the selected score range defined from cumulative distribution is shown above the corresponding column. Note the difference between the groups within the damage score 4. n ≥ 5 rats per group, N ≥ 500 glomeruli per group. A Kolmogorov-Smirnov test was used to identify significant differences between the groups (OriginPro 9.0) with a P < 0.05, distributions were significantly different. www.nature.com/scientificreports/ Other likely AEA target candidates such as GPR55, MAGL, ABHD6, and TRPV1 have only recently been recognized to explain the effects, or lack, thereof in this study. It may be possible that TRPV1, GPR18, or GPR55 could be activated to promote changes in the systemic and regional vasculature either alone or in concert with the non-CB1 receptors [72][73][74][75] . It was shown that in dissociated nodose ganglion neurons, AEA produced robust calcium influx through TRPV1 channels 75 . Methanandamide, a stable analog of AEA, potentiated the mechanosensitivity of mucosal bladder afferents, and the effect of mAEA switched from excitatory to inhibitory in the presence of TRPV1 antagonist 76 . CB1 receptors exhibited substantial neuroprotection through the diminished intracellular calcium flux. WIN 55,212-2, another CB1 receptor ligand, inhibited the NMDA-induced increase in intracellular calcium concentration 77 . However, in our studies, the application of AEA did not produce calcium flux in podocytes of freshly isolated glomeruli from SS rats on a NS diet (Supplementary Fig. S5).
A variety of signal transduction pathways are implicated in AEA-induced kidney injury, including the β-arrestin signaling pathway and cannabinoid receptors. β-arrestin 1 is responsible for signal transduction in the CB1 and CB2 receptor intracellular pathway 20,21,78 . In our study, we measured β-arrestin 1 expression in the AEA-treated group. However, neither β-arrestin 1 nor its phosphorylated form were altered during the treatment. Li et al. demonstrated that CB2 antagonist AM630 increased collagen deposition and expression levels of TGF-β1 and Smad3 during skin wound repair in the mouse 79 . In primary culture of adult human fibroblasts or in myofibroblasts stimulation with TGF-β increases CB1 receptor expression 44,49,80 . Nrf2 has also been shown to protect against renal fibrosis by inhibiting TGF-β signaling 81 . The stimulation of CB2 signaling by AM1241 diminished the development of myocardial fibrosis during the post-myocardial infarction phase via enhancing the translocation of the fibrogenesis-associated transcription factor Nrf2 to the nucleus and blocking the TGF-β1/Smad3 pathway 82 .
Here we provide evidence of the negative impact of chronic cannabinoid use in the development of kidney damage, especially in hypertensive and CKD patients. Overall, our study shows that prolonged treatment with a high dose of AEA contributes to the aggravation of SS hypertension and a consequent kidney injury, which in turn activates Nrf2-modulated TGF-β1/Smad3 pro-fibrotic signaling 79,82 . We further hypothesize that the Smad complexes translocate to the nucleus and mediate transcription of the genes that contribute to fibrosis, as previously reported 81 . Given that CB receptors are members of the GPCR family, they could also modulate the pro-fibrotic signaling pathway through an alternative TGF-β1 independent pattern 27,29,70,83,84 . Additional research on P450s that directly affect AEA to produce HETE and EET ethanolamides should be conducted in conditions of SS hypertension. It would be of interest to apply the knowledge on the AEA metabolites toward our understanding of the deleterious mechanism of CB receptor activation in the development of fibrosis, because many P450 isoforms have been found to be down-regulated during chronic liver disease 85,86 .

Material and methods
Animals and protocols. Eight-week-old male Dahl Salt Sensitive rats (SS; SS/JrHsdMcwi) were provided a normal (0.4% NaCl, # D113755, Dyets Inc.; NS) or a high salt (8% NaCl, # D100078, Dyets Inc.; HS) diets, and water ad libitum. For the surgical procedures, rats were anesthetized with inhalation of 2.5% isoflurane in 0.5 l/ min O 2 /N 2 flow. Catheters (#RPT080 Braintree, MA) were implanted in the femoral artery and vein, tunneled subcutaneously, and exteriorized at the back of the neck in a lightweight tethering spring. Following recovery from anesthesia, all rats were placed into individual cages. The catheters were connected to a pressure transducer (#041516504A, Argon Medical Devices, TX) via swivels (#375/D/22, Instech, PA) for arterial blood pressure acquisition and daily i.v. bolus drug infusion. This approach allows the collection of blood samples in conscious animals during the study. The rats were allowed to recover for at least 3 days following surgery. Anandamide (AEA, Cayman chemical, #90,050) was used at a dose 0.05 mg/kg (low dose) or 3 mg/kg (high dose). Rats were treated with AEA or a corresponding vehicle every morning for the whole duration of the experiment. To induce hypertension rats were switched from a NS to a HS diet for 14 days. Urine and blood samples were collected on day 7 and 14 on HS. Treatment with AEA was initiated from day 0 (NS, no treatment). The animal use and welfare adhered to the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals following protocols reviewed and approved by the Medical College of Wisconsin Institutional Animal Care and Use Committee. All experiments were carried out in accordance with relevant guidelines and regulations and in compliance with the ARRIVE guidelines 87 .

Kidney isolation and glomerular analysis.
By the end of the study, kidney tissue was harvested and weighted. Rats were anesthetized, and their kidneys were flushed with phosphate buffered saline (PBS) via aortic catheterization (3 ml/min/kidney until blanched). For each rat, one kidney was used for glomeruli isolation, and the other kidney coronal sections were used for Western blot. Rat kidneys were formalin fixed, paraffin embedded, sectioned, and mounted on slides. Slides were stained with Masson's trichrome stain 88 . A glomerular injury score was blindly assessed using a 0-4 scale, where 0 = no damage. A cumulative distribution plot was generated in Origin Pro 9.0 (OriginLab, Northampton, MA) using obtained glomerular injury scores, and the probability for a corresponding score interval was calculated. Medullary protein cast analysis was performed using a color thresholding method involving Metamorph software (Molecular Devices, Sunnyvale, CA). Cortical fibrosis was blindly assessed using color deconvolution in the Fiji image application (ImageJ 1. 47v, NIH).
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